Synthetic molecules that specifically react with target sequences

ABSTRACT

The present invention features biarsenical molecules. Target sequences that specifically react with the biarsenical molecules are also included. The present invention also features kits that include biarsenical molecules and target sequences. Tetraarsenical molecules are also featured in the invention.

This invention was made with Government support under Grant No. NS27177,awarded by the National Institutes of Health. The Government has certainrights in this invention.

FIELD OF THE INVENTION

This invention relates to compositions and methods for labelingmolecules, particularly small, synthetic molecules that can specificallyreact with target sequences.

BACKGROUND OF THE INVENTION

Many techniques in the biological sciences require attachment of labelsto molecules, such as polypeptides. For example, the location of apolypeptide within a cell can be determined by attaching a fluorescentlabel to the polypeptide.

Traditionally, labeling has been accomplished by chemical modificationof purified polypeptides. For example, the normal procedures forfluorescent labeling require that the polypeptide be covalently reactedin vitro with a fluorescent dye, then repurified to remove excess dyeand/or any damaged polypeptide. Using this approach, problems oflabeling stoichiometry and disruption of biological activity are oftenencountered. Furthermore, to study a chemically modified polypeptidewithin a cell, microinjection can be required. This can be tedious andcannot be performed on a large population of cells.

Thiol- and amine-reactive chemical labels exist and can be used to labelpolypeptides within a living cell. However, these chemical labels arepromiscuous. Such labels cannot specifically react with a particularcysteine or lysine of a particular polypeptide within a living cell thathas numerous other reactive thiol and amine groups.

A more recent method of intracellular labelling of polypeptides inliving cells has involved genetically engineering fusion polypeptidesthat include green fluorescent protein (GFP) and a polypeptide ofinterest. However, GFP is limited in versatility because it cannotreversibly label the polypeptide. The ability to generate a wide rangeof specifically labeled molecules easily and reliably would beparticularly useful.

SUMMARY OF THE INVENTION

In a first aspect, the invention features a biarsenical molecule of thefollowing formula:

and tautomers, anhydrides, and salts thereof;wherein:each X¹ or X², independently, is Cl, Br, I, OR^(a), or SR^(a),orX¹ and X² together with the arsenic atom form a ring having the formula

R^(a) is H, C₁-C₄ alkyl, CH₂CH₂OH, CH₂COOH, or CN;Z is 1,2-ethanediyl, 1,2-propanediyl, 2,3-butanediyl, 1,3-propanediyl,1,2 benzenediyl, 4-methyl-1,2-benzenediyl, 1,2-cyclopentanediyl,1,2-cyclohexanediyl, 3-hydroxy-1,2-propanediyl, 3-sulfo-1,2-propanediyl,or 1,2-bis(carboxy)-1,2-ethanediyl;Y¹ and Y², independently, are H or CH₃;orY¹ and Y², together form a ring such that the biarsenical molecule hasthe formula

where M is O, S, CH₂, C(CH₃)₂, or NH;R¹ and R², independently, are OR^(a), OAc, NR^(a)R^(b), or H;R³ and R⁴, independently, are H, F, Cl, Br, I, OR^(a), or R^(a);orR¹ together with R³, or R² together with R⁴, or both, form a ring inwhich

(i). one of R¹ or R³ is C₂-C₃ alkyl and the other is NR^(a) and

(ii). one of R² and R⁴ is C₂-C₃ alkyl and the other is NR^(a);

R^(b) is H, C₁-C₄ alkyl, CH₂CH₂OH, CH₂COOH, or CN;

Q is CR^(a)R^(b), CR^(a)OR^(b), C═O, or a spirolactone having theformula:

wherein the Spiro linkage is formed at C₁.

Particularly preferred is a biarsenical molecule where X¹ and X²together with the arsenic atom form a ring having the formula

Also preferred is a biarsenical where X¹ and X² together with thearsenic atom form a ring having the formula

In another preferred embodiment of the biarsenical molecule, Q is chosenfrom the following spirolactones:

A more preferred embodiment is a biarsenical where Q is

A particularly preferred biarsenical molecule has the following formula:

The tautomers, anhydrides and salts of the biarsenical molecule offormula (III) are also included.

Preferably, the biarsenical molecule specifically reacts with a targetsequence to generate a detectable signal, for example, a fluorescentsignal.

The biarsenical molecule preferably is capable of traversing abiological membrane. The biarsenical molecule preferable includes adetectable group, for example a fluorescent group, luminescent group,phosphorescent group, spin label, photosensitizer, photocleavablemoiety, chelating center, heavy atom, radioactive isotope, isotopedetectable by nuclear magnetic resonance, paramagnetic atom, andcombinations thereof.

For some applications, the biarsenical molecule can be immobilized on asolid phase, preferably by covalent coupling.

In another aspect, the invention features a kit. The kit includes theabove-described biarsenical molecule and a bonding partner that includesa target sequence. The target sequence includes one or more cysteinesand is capable of specifically reacting with the biarsenical molecule.Preferably, the target sequence includes four cysteines. The targetsequence preferably is a Cys-Cys-X-Y-Cys-Cys (SEQ ID NO:5) α-helicaldomain, where X and Y are amino acids. Preferably, X and Y are aminoacids with high α-helical propensity. In some embodiments, X and Y arethe same amino acid. In other embodiments, X and Y are different aminoacids. In particularly preferred embodiments, the target sequence is SEQID NO. 1 or SEQ ID NO. 4.

The bonding partner can include a carrier molecule, for example acarrier polypeptide. In some embodiments, the target sequence isheterologous to the carrier polypeptide. In one preferred embodiment,the target sequence specified by SEQ ID NO. 4 is linked by a peptidebond to the carboxy terminal Lys-238 in the cyan mutant of the greenfluorescent protein.

In yet another aspect, the invention features a kit that includes theabove-described biarsenical molecule and a vector that includes anucleic acid sequence encoding a target sequence. The target sequenceincludes one or more cysteines and is capable of specifically reactingwith the biarsenical molecule. Preferably, the target sequence includesfour cysteines.

In some preferred embodiments, the vector in the kit includes a nucleicacid sequence encoding a carrier polypeptide and a nucleic acid sequenceencoding a target sequence. In some embodiments, the carrier polypeptideis heterologous to the target sequence.

In another aspect, the invention features a complex. The complexincludes the above-described biarsenical molecule and a target sequence.In some preferred embodiments, the target sequence is SEQ ID NO. 1 orSEQ ID NO. 4. Preferably, the biarsenical molecule is biarsenicalmolecule of formula (III).

In another aspect, the invention features a tetraarsenical molecule. Thetetraarsenical molecule includes two biarsenical molecules of theabove-described formula. The two biarsenical molecules are coupled toeach other through a linking group. In some embodiments, thetetraarsenical molecules have formula VI, VII, or VIII.

“Bonding partner” as used herein refers to a molecule that contains atleast the target sequence.

“Heterologous” as used herein refers to two molecules that are notnaturally associated with each other.

“Associated” as used herein includes association by covalent, as well asby non-covalent interactions.

The invention provides biarsenical molecules that can be engineered toexhibit a variety of properties. For example, the biarsenical moleculecan be fluorescent. It can have different wavelengths of excitation andemission, e.g., visible or infrared. The biarsenical moleculespecifically reacts with the cysteine-containing target sequence. Inaddition, the relatively small size of both the biarsenical molecule andthe target sequence is particularly advantageous.

Other features and advantages of the invention will be apparent from thefollowing detailed description and from the claims.

SEQUENCE ID NUMBERS SEQ ID No. 1:acetyl-Trp-Glu-Ala-Ala-Ala-Arg-Glu-Ala-Cys-Cys-Arg-Glu-Cys-Cys-Ala-Arg-Ala-amideComments: The N-terminus is acetylated and the C-terminus is amidated.SEQ ID No. 2: 5′-CGG CAA TTC TTA GGC CCT GGC GCA GCA CTCCCT GCA GCA GGC CTC CCT GGC GGC GGC CTC GGC CTTGTA CAG CTC GTC CAT GCC C-3′ SEQ ID No. 3:5′-CGC GGA TCC GCC ACC ATG CAT GAC CAA CTGACA TGC TGC CAG ATT TGC TGC TTC AAA GAA GCC TTC TCA TTA TTC-3′.SEQ ID No. 4: Ala-Glu-Ala-Ala-Ala-Arg-Glu-Ala-Cys-Cys-Arg-Glu-Cys-Cys-Ala-Arg-Ala

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 illustrates pairs of biarsenical molecules that are tautomers,salts or anhydrides of each other.

FIG. 2 is a reaction scheme for the synthesis of tetraarsenicalmolecules (VI) and (VII).

FIG. 3 is a reaction scheme showing the synthesis of the biarsenicalmolecule having formula (III). The figure also illustrates the specificreaction of the biarsenical molecule (III) with the target sequence (SEQID NO:5).

FIG. 4 is a plot of the excitation and emission spectra of thebiarsenical molecule (III)/target sequence complex.

FIG. 5 is a plot of fluorescence intensity versus time in experimentswith live HeLa cells. Hela cells were either nontransfected ortransfected with the gene for the cyan mutant of green fluorescentprotein fused to the target sequence. The HeLa cells were incubated withthe biarsenical molecule (III).

FIG. 6 illustrates biarsenical molecules with detectable groups.

FIG. 7 illustrates the structure of a tetraarsenical molecule (VIII).

FIG. 8 illustrates biarsenical molecules with detectable groups.

FIG. 9 illustrates biarsenical molecules with detectable groups.

FIG. 10 illustrates a biarsenical molecule in which the fluorescentsignal is sensitive to local solvent polarity.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Biarsenical Molecule

The invention provides biarsenical molecules having the formuladescribed above in the Summary of the Invention. The present inventionalso includes tautomers, anhydrides and salts of the biarsenicalmolecule. FIG. 1 illustrates exemplary pairs of biarsenical moleculesthat are tautomers, anhydrides or salts of each other.

A number of dithiols may be used for bonding the arsenics. The dithiolgroups may protect the biarsenical molecule from reacting with lowaffinity sites, for example, single cysteine residues or dihydrolipoicacid moieties. The dithiol may form a five- or six-membered ring withthe arsenic. Vicinal dithiols that form five membered rings arepreferable. Typically, the five-membered rings may be more stable.1,3-dithiols forming six-membered rings may also be used.

The dithiol may contain additional substituents to control volatility,water solubility, proton ionization constants, redox potential, andtendency to complex with the arsenic. Increasing the molecular weightmay decrease volatility and odor. Polar substituents such ashydroxymethyl, carboxyl and sulfo decrease volatility and increase watersolubility. However, these substituents may also decrease the ability ofthe biarsenical molecule to traverse a biological membrane. Dithiolsthat contain rings may increase the affinity of the dithiol to thearsenic by organizing the two thiol groups to be in a cis-conformationready to form an additional ring with the arsenic. Examples of dithiolrings are 1,2-benzenedithiol and 1,2-cyclohexanedithiol.

Preferably, each arsenic in the biarsenical molecule is bonded to adithiol, such as 1,2-ethanedithiol (EDT). An unexpected advantage of thebiarsenical molecule of formula (III) that is bonded to EDT is that itis essentially completely nonfluorescent. Biarsenical molecules thathave detectable fluorescence are also within the scope of thisinvention.

“Q” in formula (I) is preferably a spirolactone. Particularly preferableis a biarsenical molecule in which Q is a bicyclic spirolactone as informula (III). The tautomers, anhydrides and salts of molecule (III) arealso within the scope of the invention.

The biarsenical molecule may be engineered to contain a variety ofdetectable groups. “Detectable group” as used herein refers to any atomor molecule that can be engineered into the biarsenical molecule to aidin the detection of the biarsenical molecule without significantlydestroying the biarsenical molecule's ability to react with a targetsequence.

The biarsenical molecule may be substituted at one or more positions toadd a signal generating detectable group. Inclusion of more than onedetectable group is also within the scope of this invention. Theselection of a detectable group may be made based on the ease of theprotocol for engineering the detectable group into the biarsenicalmolecule, and on the end use of the biarsenical molecule. Examples ofdetectable groups include fluorescent groups, phosphorescent groups,luminescent groups, spin labels, photosensitizers, photocleavablemoieties, chelating centers, heavy atoms, radioactive isotopes, isotopesdetectable by nuclear magnetic resonance, paramagnetic atoms, andcombinations thereof. FIGS. 6, 8 and 9 illustrate biarsenical moleculeswith some of above-mentioned detectable groups. FIG. 10 illustrates abiarsenical molecule in which the fluorescent signal is sensitive tolocal solvent polarity.

Typically, a detectable group generates a detectable signal that can bereadily monitored. Examples of detectable signals that can be monitoredinclude fluorescence, fluorescence anisotropy, time-resolvedluminescence, phosphorescence amplitude and anisotropy, electron spinresonance (ESR), singlet oxygen production, hydroxy radical-mediatedprotein inactivation, metal-ion sensing, X-ray scattering,radioactivity, nuclear magnetic resonance spectroscopy of the attachedisotope, and enhanced relaxivity of protons in the immediate vicinity ofa paramagnetic species.

Other modifying groups that aid in the use of the biarsenical moleculemay also be incorporated. For example, the biarsenical molecule may besubstituted at one or more positions to add a solid phase binding groupor a cross-linking group. The biarsenical molecule may be coupled to asolid phase.

The biarsenical molecule preferably is capable of traversing abiological membrane. The small size of the biarsenical molecule cancontribute toward the ability of the biarsenical molecule to traverse abiological membrane. Biarsenical molecules of less than 800 Daltons arepreferable for membrane traversal.

The polarity of the biarsenical molecule can also determine the abilityof the biarsenical molecule to traverse a biological membrane.Generally, a hydrophobic biarsenical molecule is more likely to traversea biological membrane. The presence of polar groups can reduce thelikelihood of a molecule to traverse a biological membrane. Abiarsenical molecule that is unable to traverse a biological membranemay be derivatized. The biarsenical molecule may be derivatized byaddition of groups that enable or enhance the ability of the biarsenicalmolecule to traverse a biological membrane. Preferably, suchderivatization of the biarsenical molecule does not significantly alterthe ability of the biarsenical molecule to subsequently react with thetarget sequence. The biarsenical molecule may also be derivatizedtransiently. In such instances, after traversing the membrane, thederivatizing group is eliminated to regenerate the original biarsenicalmolecule. Examples of derivatization methods that increase membranetraversability include esterification of phenols, ether formation withacyloxyalkyl groups, and reduction of chromophores to uncharged leucocompounds.

In some embodiments, the biarsenical molecule may be nearly orcompletely undetectable until it specifically reacts with a targetsequence. The present inventors have surprisingly discovered that thebiarsenical molecule (III) is nonfluorescent even though it issynthesized from a fluorescent molecule (parent fluorescein). Thebiarsenical molecule (III) specifically reacts with a target sequence toform a biarsenical molecule (III)/target sequence complex that isfluorescent. Moreover, the fluorescent signal generated by this complexis red-shifted by about 20 nm relative to fluorescein. This biarsenicalmolecule can be particularly useful because it provides a means tospecifically and accurately detect the presence of the biarsenicalmolecule/target sequence complex with very little background signal.

Also within the scope of this invention is a biarsenical molecule thatmay be detectable before and after it specifically reacts with a targetsequence to form the biarsenical molecule/target sequence complex. Insuch instances, it is preferable if the signal of the biarsenicalmolecule can be differentiated from the signal of the complex. Forexample, if the detectable signal of the biarsenical molecule is afluorescent signal, it would be preferable if the fluorescence of thecomplex is red-shifted or blue-shifted relative to the biarsenicalmolecule alone.

The biarsenical molecule may also lack a detectable signal, both beforeand even after specifically reacting with a target sequence. Thesebiarsenical molecules can be useful in many techniques that do notrequire a detectable signal, or that use other methods of detection.These biarsenical molecules may be useful when the goal is to attach apolypeptide to a solid substrate, cross-link two polypeptides orencourage a polypeptide domain to become α-helical.

Each of the two trivalent arsenics in the biarsenical molecule may reactwith a pair of adjacent cysteines. Thus, the biarsenical molecule mayspecifically react with four cysteines arranged in an appropriateconfiguration.

A particularly useful advantage of the specific reaction between thebiarsenical molecule and a target sequence is the reversibility of thereaction. A complex containing the biarsenical molecule and the targetsequence may be dissociated. Dissociation may be accomplished byproviding an excess of reagents such as EDT as discussed in Example 2below or other similar dithiols.

In general, the biarsenical molecule can be prepared by a shortsynthesis. FIG. 3 shows the synthesis of the biarsenical molecule (III)from commercially available fluorescein mercuric acetate (FMA).Replacement of the two mercury atoms by arsenic can be catalyzed bypalladium diacetate. The resulting 4′,5′-bis-dichloroarsine fluoresceinneed not be isolated but may be coupled directly with EDT. Biarsenicalmolecule (III) can then be purified on silica gel.

“Tetraarsenical” molecules as used herein refer to molecules thatcontain four arsenics. In some embodiments, tetraarsenical molecules aretwo biarsenical molecules chemically coupled to each other through alinking group. Tetraarsenical molecules may be synthesized in a varietyof ways. FIG. 2 illustrates one scheme for synthesizing tetraarsenicalmolecules that have two biarsenical molecules coupled through either apara- or a meta-dicarboxylbenzene. The synthesis in FIG. 2 results intwo types of molecules, a meta- and a para-substituted tetraarsenicalmolecule. FIG. 7 is another example of a tetraarsenical molecule coupledthrough a dialkylamido linking group. Other suitable linking groupsinclude phenyl, napthyl, and biphenyl groups. It follows that thetetraarsenical molecule can react with two target sequences.Tetraarsenical molecules may be particularly useful as cross-linkingagents, e.g. intra-molecular and intermolecular cross-linking agents.

Target Sequence

Generally, the target sequence includes one or more cysteines,preferably four, that are in an appropriate configuration for reactingwith the biarsenical molecule. The target sequence alone may be able toreact with the biarsenical molecule. The target sequence can vary insize. Typically it contains at least 6 amino acids. Preferably, thetarget sequence is at least 10 amino acids. Alternatively, the targetsequence may only adopt an appropriate configuration when it isassociated with a carrier molecule. For example, the biarsenicalmolecule may react with a target sequence only when the target sequenceis placed in an α-helical domain of a polypeptide.

The target sequence may have an amino acid sequence such that two pairsof cysteines are arranged to protrude from the same face of an α-helix.Preferably, the four sulfurs of the cysteines form a parallelogram.

The target sequence alone may not be completely helical under thereaction conditions. For example, reaction of a first arsenic with apair of cysteines may nucleate an α-helix and position the two othercysteines favorably for reacting with the other arsenic of thebiarsenical molecule.

The secondary structure of the target sequence may be an α-helix. Anα-helical target sequence may include a primary amino acid sequence ofCys-Cys-X-Y-Cys-Cys (SEQ ID NO:5). The cysteines in this primary aminoacid sequence are positioned for encouraging arsenic interaction acrosshelical turns. The four cysteine residues of this sequence contain thesulfurs that specifically react with the biarsenical molecule. In thissequence, X and Y may be any amino acid, including cysteine. In someembodiments, X and Y may be the same amino acid and in otherembodiments, X and Y may be different amino acids. The use of naturalamino acids is preferable. Preferable amino acids at positions X and Yare amino acids with high α-helical propensity. Amino acids that havehigh α-helical propensity include alanine, leucine, methionine, andglutamate.

Formation of an α-helix may also be favored by incorporation ofoppositely charged amino acids that are separated by about three aminoacids. These oppositely charged amino acids may be properly placed toform salt bridges across one turn of an α-helix. An example of a pair ofoppositely charged amino acids is arginine and glutamate. Merutka &Stellwagen., Biochemistry 30: 1591-1594 and 4245-4248 (1991). It ispreferable to position glutamate toward the N-terminus of the α-helixand arginine toward the C-terminus for favorable interaction with thedipole of an α-helix. The N-terminus of the target sequence may beacetylated. The C-terminus of the target sequence may be amidated.

A target sequence containing other secondary structures is also withinthe scope of this invention. For example, the one or more cysteines ofthe target sequence may be within a β-sheet structure. Other secondarystructures are possible as long as the target sequence can react withthe biarsenical molecule.

An example of a target sequence is SEQ ID NO. 1, as well as variantsthereof that retain reactivity with the biarsenical molecule. In thistarget sequence, the N-terminus is acetylated and the C-terminus isamidated. A target sequence that is not acetylated and amidated at theN- and C-terminus is also within the scope of this invention. “Variant”target sequences contain one or more amino acid substitutions, typicallywith amino acid substitutes of approximately the same charge andpolarity. Such substitutions can include, e.g., substitutions within thefollowing groups: valine, isoleucine, leucine, methionine; asparticacid, glutamic acid; asparagine, glutamine; serine, threonine; lysine,arginine; and phenylalanine, tyrosine. In general, such substitutions donot significantly affect the function of a polypeptide. Methods forproducing target sequences include molecular biology methods andchemical polypeptide synthesis methods.

Bonding Partner

The bonding partner includes a cysteine-containing target sequence thatspecifically reacts with the biarsenical molecule. In addition to thetarget sequence, the bonding partner may also include a carrier moleculethat is associated with the target sequence. Examples of carriermolecules include polypeptides, nucleic acids, sugars, carbohydrates,lipids, natural polymers, synthetic polymers, and other biologically orchemically active molecules.

Polypeptide Bonding Partner

In some embodiments, the carrier molecule can be a polypeptide. In suchcases, the polypeptide is referred to as a carrier polypeptide. In theseembodiments, the bonding partner includes the carrier polypeptide thatis associated with the target sequence. A “polypeptide bonding partner”as used herein refers to a bonding partner that includes a carrierpolypeptide and a target sequence. The carrier polypeptide can be anypolypeptide of interest. Examples of carrier polypeptides includeantibodies, receptors, hormones, enzymes, binding proteins, andfragments thereof.

The target sequence and the carrier polypeptide may be associated witheach other covalently. Alternatively, the carrier polypeptide and thetarget sequence may be non-covalently associated.

The position of the target sequence with respect to the carrierpolypeptide can vary in a bonding partner. The target sequence may beattached to the C-terminal end of the carrier polypeptide.Alternatively, the target sequence may be attached to the N-terminal endof the carrier polypeptide.

The target sequence may also be internal to the carrier polypeptide. Aninternal target sequence may be produced by inserting the targetsequence at an internal site in the carrier polypeptide. Alternatively,an internal target sequence may be created by modifying one or moreamino acids of the polypeptide to create a target sequence. Suchinternal sites are typically selected for their α-helical structures.Computer algorithms and x-ray crystallography data can be used toidentify α-helical structures within polypeptides.

In some embodiments, the target sequence and the carrier polypeptide areheterologous to each other. The carrier polypeptide and the targetsequence are also heterologous if the amino acid sequence of the carrierpolypeptide is altered at one or more amino acid positions to generatethe target sequence.

Any of the polypeptides and/or target sequences used in the invention,collectively referred to herein as “polypeptides”, can be synthesized bysuch commonly used methods as t-BOC or FMOC protection of α-aminogroups. Both methods involve stepwise syntheses whereby a single aminoacid is added at each step starting from the C terminus of the peptide(See, Coligan, et al., Current Protocols in Immunology, WileyInterscience, 1991, Unit 9). Polypeptides may also be synthesized by thewell known solid phase peptide synthesis methods described inMerrifield, (J. Am. Chem. Soc., 85:2149, 1962), and Stewart and Young,Solid Phase Peptides Synthesis, (Freeman, San Francisco, 1969, pp.27-62), using a copoly(styrene-divinylbenzene) containing 0.1-1.0 mMolamines/g polymer. On completion of chemical synthesis, the polypeptidescan be deprotected and cleaved from the polymer by treatment with liquidHF-10% anisole for about ¼-1 hours at 0° C. After evaporation of thereagents, the polypeptides are extracted from the polymer with 1% aceticacid solution which is then lyophilized to yield the crude material.This can normally be purified by such techniques as gel filtration onSephadex G-15 using 5% acetic acid as a solvent. Lyophilization ofappropriate fractions of the column will yield the homogeneouspolypeptide or polypeptide derivatives, which can then be characterizedby such standard techniques as amino acid analysis, thin layerchromatography, high performance liquid chromatography, ultravioletabsorption spectroscopy, molar rotation, solubility, and quantitated bythe solid phase Edman degradation.

Polypeptides may also be produced by the “native chemical” ligationtechnique which links together polypeptides (Dawson et al., Science,266:776, 1994). Protein sequencing, structure and modeling approachesfor use with a number of the above techniques are disclosed in ProteinEngineering, loc. cit., and Current Protocols in Molecular Biology,Vols. 1 & 2, supra.

The polypeptides can also be non-polypeptide compounds that mimic thespecific reaction and function of a polypeptide (“mimetics”). Mimeticscan be produced by the approach outlined in Saragovi et al., Science,253:792-795 (1991). Mimetics are molecules which mimic elements ofpolypeptide secondary structure. See, for example, Johnson et al.,“Peptide Turn Mimetics”, in Biotechnology and Pharmacy, Pezzuto et al.,Eds., (Chapman and Hall, New York 1993). The underlying rationale behindthe use of peptide mimetics is that the peptide backbone exists chieflyto orient amino acid side chains in such a way as to facilitatemolecular interactions. For the purposes of the present invention,appropriate mimetics can be considered to be the equivalent of any ofthe polypeptides used in the invention.

Vector

Useful polypeptides may also be generated by nucleic acid techniquesinvolving expression of nucleic acid sequences that encode thepolypeptides. The term “vector” refers to a plasmid, virus or othervehicle known in the art that has been manipulated by insertion orincorporation of a nucleic acid sequence.

Methods that are well known in the art can be used to construct vectors,including in vitro recombinant DNA techniques, synthetic techniques, andin vivo recombination/genetic techniques. (See, for example, thetechniques described in Maniatis et al. 1989 Molecular Cloning ALaboratory Manual, Cold Spring Harbor Laboratory, N.Y.)

Suitable vectors include T7-based expression vectors for expression inbacteria (Rosenberg, et al., Gene, 56:125, 1987), the pMSXND expressionvector for expression in mammalian cells (Lee and Nathans, J. Biol.Chem., 263:3521, 1988) and baculovirus-derived vectors for expression ininsect cells. Retroviral vectors may also be used.

Examples of retroviral vectors include Moloney murine leukemia virus,(MoMuLV), Harvey murine sarcoma virus (HaMuS-V), murine mammary tumorvirus (MuMTV), and Rous Sarcoma Virus (RSV). Expression vectors suitablefor in vitro expression may also be used.

Generally, the vector includes a nucleic acid sequence encoding thetarget sequence. Typically, the nucleic acid sequence is a DNA sequence,although the nucleic acid can be an RNA sequence. The nucleic acidsequence can be any sequence that encodes a target sequence capable ofreaching with the biarsenical molecule. This can include nucleic acidsequences that are degenerate variants of each other. By “degeneratevariants” is meant nucleic acid sequences that encode the same aminoacid sequence, but in which at least one codon in the nucleotidesequence is different. Degenerate variants occur due to the degeneracyof the genetic code, whereby two or more different codons can encode thesame amino acid. Nucleic acid sequences of the present invention may besynthetic.

The vector may also contain a nucleic acid sequence encoding a carrierpolypeptide, in addition to the nucleic acid sequence encoding thetarget sequence. Nucleic acid sequences encoding the carrier polypeptideand the target sequence can form a recombinant gene that, whenexpressed, produces a polypeptide bonding partner.

The nucleic acid sequence encoding the target sequence can be on the 5′or 3′-end of the nucleic acid sequence encoding the carrier polypeptide.Alternatively, the nucleic acid sequence encoding the target sequencecan be internal to the nucleic acid sequence encoding the carrierpolypeptide. In such a case, the nucleic acid sequence encoding thetarget sequence can be spliced into an internal site of the nucleic acidsequence encoding the carrier polypeptide. In this case, the nucleicacid sequence encoding the target sequence is flanked by nucleic acidsequences encoding the carrier polypeptide.

The nucleic acid sequence encoding the carrier polypeptide may containan appropriate restriction enzyme site within its nucleic acid sequencethat can be used for inserting the nucleic acid sequence encoding thetarget sequence. Alternatively, an appropriate restriction enzyme sitecan be engineered in the nucleic acid sequence encoding the carrierpolypeptide at a desired location. A restriction enzyme site may beengineered by any number of known methods.

The nucleic acid sequence encoding the carrier polypeptide may byaltered at one or more positions to generate the nucleic acid sequencethat encodes the target sequence. For example, calmodulin can be alteredto create a target sequence as described in Example 3. In someembodiments, changes in the nucleic acid sequence encoding the carrierpolypeptide may be made to generate a nucleic acid encoding a targetsequence without substantially affecting the function of the carrierpolypeptide.

Site-specific and region-directed mutagenesis techniques, as well asstandard recombinant techniques can be employed for generating some ofthe nucleic acid sequences that encode the polypeptides used in theinvention. See Current Protocols in Molecular Biology, Vol. 1, Ch. 8(Ausubel et al., eds., J. Wiley & Sons 1989 & Supp. 1990-93); ProteinEngineering (Oxender & Fox eds., A. Liss, Inc. 1987). In addition,linker-scanning and PCR-mediated techniques can be employed formutagenesis. See PCR Technology (Erlich ed., Stockton Press 1989);Current Protocols in Molecular Biology, Vols. 1 & 2, supra.

The vector may also contain any number of regulatory elements fordriving expression of the polypeptides. Nucleic acid sequences encodingpolypeptides may be operatively associated with a regulatory element.Regulatory elements include, but are not limited to, inducible andnon-inducible promoters, enhancers, operators and other elements thatdrive or otherwise regulate gene expression.

Typically, a nucleic acid sequence encoding a polypeptide is operativelylinked to a promoter that is active in the appropriate environment, i.e.a host cell. A variety of appropriate promoters are known in the art andmay be used in the present invention. The promoter may be a promoterthat naturally drives expression of the carrier polypeptide. Thepromoter may be a viral promoter, a bacterial promoter, a yeastpromoter, insect promoter or a plant promoter, and can be hostcell-specific. Examples of promoters include, without limitation, T7,metallothionein I, or polyhedron promoters. For example, if thepolypeptides will be expressed in a bacterial system, induciblepromoters such as pL of bacteriophage gamma, plac, ptrp, ptac (trp-lachybrid promoter) and the like may be used. In mammalian cell systems,promoters derived from the genome of mammalian cells (e.g.,metallothionein promoter) or from mammalian viruses (e.g., theretrovirus long terminal repeat; the adenovirus late promoter; thevaccinia virus 7.5K promoter) may be used. Promoters produced byrecombinant DNA or synthetic techniques may also be used.

The vector may also include enhancer sequences. Enhancer sequences canbe placed in a variety of locations in relation to polypeptide-encodingnucleic acid sequences. For example, enhancer sequences can be placedupstream or downstream of the coding sequences, and can be locatedadjacent to, or at a distance from, the polypeptide encoding nucleicacid sequences.

The vector may also contain a nucleic acid sequence encoding aselectable marker for use in identifying host cells containing a vector.A selectable marker in a vector typically confers some form of drug orantibiotic resistance to the host cells carrying the vector.

A number of selection systems may be used. In bacterial host cells, anumber of antibiotic markers may be used. Antibiotic markers includetetracycline, ampicillin, and kanamycin. In mammalian host cells,selections systems include, but are not limited to herpes simplex virusthymidine kinase (Wigler et al., 1977, Cell 11:223),hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski,1962, Proc. Natl. Acad. Sci. USA 48: 2026), and adeninephosphoribosyltransferase (Lowy, et al., 1980, Cell 22: 817). Also,antimetabolite resistance can be used as the basis of selection fordhfr, which confers resistance to methotrexate (Wigler, et al., 1980,Proc. Natl. Acad. Sci. USA 77: 3567; O'Hare, et al., 1981, Proc. Natl.Acad. Sci. USA 78: 1527); gpt, which confers resistance to mycophenolicacid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072; neo,which confers resistance to the aminoglycoside G-418 (Colberre-Garapin,et al., 1981, J. Mol. Biol. 150: 1); and hygro, which confers resistanceto hygromycin (Santerre, et al., 1984, Gene 30: 147) genes. Additionalselectable genes include, trpB, which allows cells to utilize indole inplace of tryptophan; hisD, which allows cells to utilize histinol inplace of histidine (Harman & Mulligan, 1988, Proc. Natl. Acad. Sci. USA85:8047); and ODC (ornithine decarboxylase) which confers resistance tothe ornithine decarboxylase inhibitor, 2-(difluoromethyl)-DL-ornithine,DFMO (McConlogue L., 1987, In: Current Communications in MolecularBiology, Cold Spring Harbor Laboratory ed.).

Host Cell

A host cell may carry an exogenous bonding partner. “Exogenous” as usedherein refers to any molecules that are introduced into a host cell. Inpreferred embodiments, the exogenous bonding partner is a polypeptidebonding partner.

A “host cell” can be any cell capable of carrying an exogenous bondingpartner. Examples of host cells include bacterial cells, yeast cells,insect cells, mammalian cells, and plant cells. A suitable host celltype includes a cell of the following types: HeLa cells, NIH 3T3(Murine), My 1 lu (Mink), BS-C-1 (African Green Monkey) and humanembryonic kidney (HEK) 293 cells. Such cells are described, for example,in the Cell Line Catalog of the American Type Culture Collection (ATCC).Cells that can stably maintain a vector may be particularlyadvantageous. See, for example, Ausubel-et al., Introduction of DNA IntoMammalian Cells, in Current Protocols in Molecular Biology, sections9.5.1-9.5.6 (John Wiley & Sons, Inc. 1995). Preferably, host cells donot naturally express polypeptides containing target sequences thatreact with molecules of the invention.

An exogenous bonding partner can be introduced into a host cell by avariety of appropriate techniques. These techniques includemicroinjection of bonding partners and expression within a cell ofnucleic acids that encode bonding partners.

A host cell can be manipulated to carry an exogenous bonding partner byintroducing a nucleic acid sequence that, when expressed, produces thebonding partner. Any of the vectors described above containing a nucleicacid sequence encoding a bonding partner may be introduced into a hostcell. A non-replicating nucleic acid molecule, such as a linear moleculethat can express a bonding partner is also within the scope of thisinvention.

The expression of a desired nucleic acid molecule may occur throughtransient expression of the introduced polypeptide-encoding nucleic acidsequence. Alternatively, permanent expression may occur throughintegration of the introduced nucleic acid sequence into a hostchromosome. Therefore the cells can be transformed stably ortransiently. The term “host cell” may also include any progeny of a hostcell. It is understood that all progeny may not be identical to theparental cell since there may be mutations that occur duringreplication. However, such progeny are included when the term “hostcell” is used.

Typically, the vector that includes the nucleic acid sequence encodingthe bonding partner is introduced into a host cell. Methods of stabletransfer, meaning that the vector having the bonding partner encodingnucleic acid sequence is continuously maintained in the host, are knownin the art. The vector, with appropriate regulatory elements forexpression in a host cell, can be constructed as described above.

The vector may be introduced into a host cell by any conventionalmethod, including retroviral transduction, electroporation, calciumphosphate co-precipitation, biolistics and liposome-based introduction.See, for example, Ausubel et al., Introduction of DNA Into MammalianCells, in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (John Wiley & Sons,Inc. 1995).

A variety of host cell-specific expression vector systems may beutilized to express polypeptides in a host cell. These includemicroorganisms such as bacteria transformed with recombinantbacteriophage DNA, plasmid DNA or cosmid DNA expression vectors; yeasttransformed with recombinant yeast expression vectors; plant cellsystems infected with recombinant virus expression vectors (e.g.,cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid); insect cell systems infected with recombinant virus expressionvectors (e.g., baculovirus); or animal cell systems infected withrecombinant virus expression vectors (e.g., retroviruses, adenovirus,vaccinia virus), or transformed animal cell systems engineered forstable expression. Polypeptides may require translational and/orpost-translational modifications such as addition of carbohydrates.These modifications can be provided by a number of systems, e.g.,mammalian, insect, yeast or plant expression systems.

Eukaryotic systems, and preferably mammalian expression systems, allowfor proper post-translational modifications of expressed mammalianpolypeptides to occur. Eukaryotic cells which possess the cellularmachinery for proper processing of the primary transcript,glycosylation, phosphorylation, and advantageously, plasma membraneinsertion of a polypeptide may be used as host cells.

Depending on the host cell and the vector system utilized, any of anumber of suitable transcription and translation elements, includingconstitutive and inducible promoters, transcription enhancer elements,transcription terminators, etc. may be used in the expression vector(see e.g., Bitter et al., 1987, Methods in Enzymology, 153:516-544) asdescribed earlier. Selection of the appropriate transcription andtranslation elements are readily apparent to a person of ordinary skillin the art.

Vectors based on bovine papilloma virus which have the ability toreplicate as extrachromosomal elements may be of particular interest(Sarver et al., 1981, Mol. Cell. Biol. 1:486). Shortly after entry ofthis DNA, the plasmid replicates to about 100 to 200 copies per cell.Transcription of the polypeptide encoding nucleic acid sequences doesnot require integration of the plasmid into the host's chromosome,thereby yielding a high level of expression. These vectors can be usedfor stable expression by including a selectable marker in the plasmid,such as, for example, the neo gene.

Factors of importance in selecting a particular expression systeminclude: the ease with which a host cell that contains the vector may berecognized and selected from a host cell that does not contain thevector; the number of copies of the vector which are desired in aparticular host cell; and whether it is desirable to be able to“shuttle” the vector between different types of host cells.

Uses of Biarsenical Molecules and Target Sequences

The biarsenical molecule, in combination with the target sequence, forma biarsenical molecule/target sequence complex that is useful in anumber of methods. The complex is particularly useful in methods forlabeling a carrier molecule. The carrier molecule can be associated withthe target sequence to form a bonding partner. The bonding partner maybe produced by any method, including a number of the above-describedmethods. In preferred embodiments, the carrier molecule is apolypeptide.

A bonding partner that includes a target sequence is contacted with thebiarsenical molecule. Contact of the biarsenical molecule with thebonding partner is performed under conditions appropriate for a specificreaction to occur between the biarsenical molecule and the targetsequence to form the biarsenical molecule/target sequence complex.

A biarsenical molecule/target sequence complex that generates adetectable signal may be used if detection of a labeled carrier moleculeis desired. A particular advantage of using the biarsenical molecule andthe target sequence for labeling is the specificity and thereversibility of the interaction. The biarsenical molecule/targetsequence complex may be dissociated, for example, after the detection ofthe complex.

The biarsenical molecule may be added to a composition that includes thetarget sequence. The biarsenical molecule may or may not be capable oftraversing a membrane. The bonding partner may be, for example, in atest tube, a microliter well or immobilized on a solid phase. Uses ofthe biarsenical molecule/target sequence complex include polypeptidepurification, immunoassays, and other biological and chemical assays.

Immobilization of either the biarsenical molecule or the bonding partnerto a solid phase may be particularly useful. Immobilization may includeadsorption, absorption or covalent bonding. A solid phase may be inertor it may be reactive for coupling. Solid phases that may be usedinclude glass, ceramics, and natural or synthetic polymeric materials.Examples of polymeric materials include cellulose-based materials,dextran-based materials, and polystyrene-based materials.

The biarsenical molecule may be contacted with a bonding partner in aliving cell. The bonding partner may be introduced into a cell orproduced within a cell. A biarsenical molecule capable of traversing abiological membrane is preferable when the biarsenical molecule isintroduced outside the cell and the bonding partner is inside the cell.Typically, a membrane traversing biarsenical molecule is preferable foruse within a living cell. Examples of uses of the biarsenicalmolecule/target sequence complex within cells include polypeptideinteractions, polypeptide location, polypeptide quantifications, nucleicacid molecule identification and location. One use of the biarsenicalmolecule of formula (III) in combination with the target sequence inHeLa cells is demonstrated in Example 2 below.

The biarsenical molecule may be used to induce a more favorableconformation of the bonding partner. For example, the bonding partnermay have two possible conformations, but one of the conformations may bemore functionally important. The bonding partner when it specificallyreacts with the biarsenical molecule may adopt the more functionallyimportant conformation. A functionally important conformation may be,for example, a conformation that can bind a drug.

A tetraarsenical molecule of the present invention can be used tocross-link two bonding partners. Each of the bonding partners includes atarget sequence. In a preferred embodiment, each bonding partnercontains a target sequence and a carrier molecule. The carrier moleculemay be a polypeptide. The polypeptides in each of the bonding partnersmay be same. Alternatively, the polypeptides in each bonding partner maybe different. The target sequences may be the same or they may bedifferent in each bonding partner. For example, cross-linking ofpolypeptides may be valuable in studying the effects of polypeptidedimerization on signal transduction. Ho S. N., Biggar S. R., Spencer D.M., Schreiber S. L., and Crabtree G. R., Nature 382: 822-826 (1996);Spencer D. M., Wandless T. J., Schreiber S. L., and Crabtree G. R.Science 262: 1019-1024 (1993). The carrier polypeptide may be an enzymeor an antibody.

In some embodiments, a bonding partner containing the target sequenceand an antibody as the carrier polypeptide may be cross-linked via atetraarsenical molecule to a bonding partner containing the targetsequence and an enzyme, as the carrier polypeptide. Such a compositionmay be useful, for example, in enzyme immunoassays.

A wide variety of assays exist that use detectable signals as a means todetermine the presence or concentration of a particular molecule.Examples of such assays include immunoassays to detect antibodies orantigens, enzyme assays, chemical assays and nucleic acid assays. Anabove described biarsenical molecule/target sequence complex can beuseful in these assays.

In general, assays may be performed as follows. A sample containing amolecule of interest associated with either the biarsenical molecule orthe target sequence may be contacted with the target sequence or thebiarsenical molecule, respectively. The resulting solution is thenmonitored for the presence of a detectable signal or a change in adetectable signal.

A particularly useful characteristic of the biarsenical molecule/targetsequence complex is that the complex may be dissociated by adding anexcess reagent such as EDT. The dissociation of the complex may beparticularly useful in assays, polypeptide purification schemes, andwithin cells.

The invention will be further understood with reference to the followingexamples, which are purely exemplary, and should not be taken aslimiting the true scope of the present invention as described in theclaims.

EXAMPLES Materials

Instruments:

UV-Vis: Cary 3E

Fluorimeter: Spex DM3000 fluorescence spectrometer with two SPEX 16810.22 m monochromators 450 W Xenon lamp.

Countercurrent: High speed counter current chromatograph (P.C. Inc.)with Shimadzu LC-8A preparative LC pump unit.

HPLC: Dionex Biol. C Column. Dionex Ionpac NSI (10-32) reverse phase.

NMR: Varian Gemini 200 MHz

Mass spectra: Hewlett-Packard 5989B electrospray mass spectrometer.

All reagents and solvents were purchased from Aldrich or Fisher and wereused as received.

Example 1 Synthesis and Characterization of Biarsenical Molecule (III)and a Target Sequence

Synthesis. A biarsenical molecule of formula (III)(4′,5′-bis(2-arsa-1,3-dithiolan-2-yl)fluorescein), herein referred to asbiarsenical molecule (III), was prepared by a short synthesis fromcommercially available fluorescein mercuric acetate (FMA). All of thesteps were conducted at room temperature, unless otherwise indicated.FMA (72 mg, 85 μmol) was suspended in 1.5 mL dry N-methylpyrrolidinoneunder argon and dissolved to a pale yellow solution upon addition of 144μl (1.7 mmol) of arsenic trichloride. A few grains of palladiumdiacetate and 120 μl dry diisopropylethylamine (DIEA) were added withstirring. After three hours, the reaction was added dropwise to asolution of 20 mL of 50% acetone:0.25 M phosphate buffer pH 7.1,2-ethanedithiol (EDT) (285 μl, 3.4 mmol) was then added followed bychloroform (20 mL) after five minutes. After 20 minutes of stirring, thereaction mixture was diluted with 100 mL water and separated. Theaqueous layer was extracted (2×20 mL) with chloroform. The combinedchloroform layers were washed (1×25 mL) with 0.1 M Na₂EDTA pH 7, driedwith Na₂SO₄ and evaporated. The resulting oil was dissolved in toluene(100 mL) and washed (3×25 mL) with water. After drying with Na₂SO₄ andevaporation, the product was purified by SiO₂ column chromatography,loaded in toluene and eluted with 10% ethylacetate-toluene. Triturationwith 95% ethanol gave an orange-red solid. The yield was 21 mg (37%).¹H-NMR (CDCl₃ with a trace of CD₃OD) results were 2.3 (br s, 2+H, OH),3.57 (m, 8H, —SCH₂CH₂S—), 6.60 (d, 2H, H-2′ J=8.8 Hz), 6.69 (d, 2H, H-1′J=8.8 Hz), 7.19 (d, 1H, H-7), 7.66 (m, 2H, H-5,6), 8.03 (d, 1H, H-4).

Solutions of the material gave a single spot with thin layerchromatography (TLC) (ethylacetate-hexane 1:1, 0.1% acetic acid, R_(f)0.55), but on aging gave more polar material. Addition of a slightexcess of EDT reversed this process suggesting some dissociation of thecomplex occurs with time. The extinction coefficient was 41,000 M⁻¹cm⁻¹at 507.5 nm in 0.1 M KCl, 10 mM KMOPS, pH 7.3. In alkaline solution (pH13), the extinction coefficient was 55,000 M⁻¹cm⁻¹ at 496.5 nm. Massspectrum analysis indicated a molecular weight of 664.0 Da compared tothe calculated molecular weight of 664.6 Da.

Target sequence synthesis. The crude polypeptideacetyl-Trp-Glu-Ala-Ala-Ala-Arg-Glu-Ala-Cys-Cys-Arg-Glu-Cys-Cys-Ala-Arg-Ala-amide(SEQ ID NO. 1), prepared by the UCSD peptide synthesis facility, waspurified by counter current chromatography on a 390 mL planetary coil(PC, Inc.) revolving at 800 RPM using n-butanol as the stationary phaseand water as the mobile phase (4 mL/min). The polypeptide eluted in abroad peak centered at 75 minutes after the water solvent front. Thistarget sequence was used in the examples below unless otherwiseindicated.Formation of target sequence/biarsenical molecule (III) complex.Biarsenical molecule (III) (3 μl of 1 mM solution in DMSO) was added to100 μl of 25 μM target sequence (SEQ. ID NO. 1) in 25 mM phosphate, pH7.4, 100 mM KCl, 1 mM mercaptoethanesulfonate. After 1.5 hours, thereaction mixture (at room temperature) was injected onto a Dionex IonPacNS1 reverse phase HPLC column, gradient 20% to 46% acetonitrile (0.1%TFA) from 3 to 17 minutes. The complex eluted at 14.7 minutes (Freetarget sequence elutes at 12.6 minutes.) Mass spectrum analysisindicated a molecular weight of 2414.99 Da and the calculated molecularweight for the 1:1 complex was 2415.33 Da.Quantum yield of target sequence/biarsenical molecule (III) complex.Solutions of fluorescein in 0.1 N NaOH and of targetsequence/biarsenical molecule (III) complex in 25 mM phosphate, pH 7.4and 100 mM KCl were adjusted to equal absorbances (0.0388) at 499 nm.The ratio of the integrated emission (excitation at 499 nm) of targetsequence/biarsenical molecule (III) complex relative to fluorescein wasmultiplied by 0.9, the quantum yield of fluorescein, giving a quantumyield 0.44 for the target sequence/biarsenical molecule (III) complex.

The biarsenical molecule (III) and the target sequence form a 1:1complex as demonstrated by electrospray mass spectroscopy. This complexhas a fluorescence quantum yield of 0.44 with excitation maximum at 508nm and emission maximum at 528 nm (FIG. 4). The complex was ofsufficient stability to remain intact in the presence of up to 100equivalents of 2,3 dimercapto-1-propanol (BAL). Incubations with BALwere done at room temperature for 15 minutes. A 100 nM solution of thebiarsenical molecule (III)/target sequence complex was barely affectedby the addition of 1 μM or 10 μM BAL. Addition of 100 μM BAL resulted ina significant reduction in fluorescence, indicating that the biarsenicalmolecule (III)/target sequence complex was cleaved.

Monothiols were required for the efficient formation of the complex.That the monothiol is not functioning solely as a reducing agent wasdemonstrated by the fact that replacing the monothiol withtriscarboxyethylphosphine does not result in efficient formation of thecomplex.

Example 2 The Use of Biarsenical Molecule (III) in Hela Cells

A polypeptide bonding partner that contains the target sequence (SEQ. IDNO. 4) attached to the cyan mutant of the green fluorescent protein wasexpressed in HeLa cells.

Expression of Cyan GFP-target sequence fusion in HeLa cells. Usingstandard molecular biology techniques, the target sequence (SEQ ID NO.4) (with the tryptophan in SEQ ID NO. 1 replaced by an alanine) wasattached to cyan fluorescent protein (CFP). CFP is the Green FluorescentProtein (GFP) of Aequorea victoria with the following additionalmutations: F64L, S65T, Y66W, N146I, M153T, V163A, N212K. Miyawaki A., etal. Nature 388:882-7 (1997). Fusion of the target sequence to theC-terminus of cyan GFP was accomplished using a PCR primer. The PCRprimer had the following oligonucleotide sequence 5′-CGG CAA TTC TTA GGCCCT GGC GCA GCA CTC CCT GCA GCA GGC CTC CCT GGC GGC GGC CTC GGC CTT GTACAG CTC GTC CAT GCC C-3′ (SEQ ID NO. 2) encoding for the expression ofthe target sequence. It was inserted into the pcDNA3 vector (Invitrogen,Carlsbad, Calif.) using HindIII and EcoRI restriction sites. Afteramplification in DH5 bacteria, it was transfected (at 37° C.) into HeLacells using the Lipofectin system from GibcoBRL.Measurement of FRET in HeLa cells. Three days after transfection, aconcentration of 1.0 μM biarsenical molecule (III) and 10.0 μMethanedithiol was applied to the transfected cells. Fluorescence changeswere observed using a 440DF20 filter (Omega Optical, Brattleboro, Vt.)and a 4% transmittance neutral density filter for excitation and 480DF30and 635DF50 filters for emission.

CFP is an engineered mutant of GFP with shorter wavelength excitationand emission maxima. It was chosen because its emission overlaps wellwith the excitation of biarsenical molecule (III)/target sequencefluorophore. The target sequence was fused to the C-terminus of CFPwithout additional linkers. The crystal structure of GFP shows that thefinal C-terminal amino acids are disordered and should thus provideenough flexibility to insure that the molecule (III)/target sequencefluorophore is not frozen in an unfavorable position for fluorescenceresonance energy transfer (FRET).

Fluorescence changes were observed upon contacting cells withbiarsenical molecule (III). A marker was used to indicate the cells thatwere expressing the target sequence and also to demonstrate thatbiarsenical molecule (III) was reacting with the target sequence in aspecific manner. Fluorescence of the CFP indicated cells were expressingthe target sequence and FRET between the CFP and biarsenical molecule(III)/target sequence demonstrated the specificity of the reaction.

HeLa cells expressing the fusion protein were contacted with biarsenicalmolecule (III) on a fluorescence microscope stage. Observed changes influorescence indicated that the desired specific reaction between thebiarsenical molecule (III) and the target sequence had occurred. FIG. 5shows a time course of the fluorescence intensity for two cells at twodifferent wavelengths (480 nm and 635 nm), corresponding to emission ofCFP and the long-wavelength tail of the emission of biarsenical molecule(III)/target sequence, as well as traces for non-transfected cells inthe same microscope field. At the start of the experiment, it can beseen that excitation of CFP resulted in emission mostly in the 480 nmchannel. Upon addition of 1.0 μM biarsenical molecule (III) mixed with10 μM EDT to inhibit background staining, the intensity of fluorescenceat 480 nm decreased as energy was transferred from the CFP to thebiarsenical molecule (III) which had reacted with the target sequence.There was a corresponding increase in fluorescence in the 635 nm channeldue to biarsenical molecule (III)/target sequence emission. Upon removalof the biarsenical molecule (III) solution, there was little change.Addition of 10 μM EDT resulted in only a small change.

Reversibility of the reaction was demonstrated by treating the cellswith 1 mM EDT, a concentration sufficient to remove biarsenical molecule(III) from the target sequence in solution. However, the removal incells was fast but not complete. Recovery of CFP fluorescence indicatedthat the former reduction of signal in this channel was indeed due toenergy transfer and not to degradation of the CFP polypeptide.

An outstanding feature in this experiment was the absence of backgroundfluorescence under the FRET conditions from either untransfected cellsin the field or from the media containing biarsenical molecule (III).This was mostly due to the nonfluorescence of biarsenical molecule (III)in the presence of excess EDT. It was also helpful that in thisexperiment biarsenical molecule (III)/target sequence could only beexcited by energy transfer as it had virtually zero excitation amplitudeat 440 nm where CFP was illuminated.

In a separate experiment, conducted under the same conditions butdifferent wavelengths, the signal at 535 nm was also investigated usingan excitation at 480 nm, corresponding roughly to the spectra ofbiarsenical molecule (III)/target sequence. At these wavelengths,untransfected cells developed about 10% of the fluorescence of cellsexpressing the CFP-target sequence fusion, after subtraction of thesignal from CFP that was present before application of biarsenicalmolecule (III). This level of background was low enough not to interferewith the use of biarsenical molecule (III) as a labeling reagent formany applications.

Example 3 Target Sequence Generated in Calmodulin

A target sequence that included the sequence Cys-Cys-X-Y-Cys-Cys (SEQ IDNO:5) was introduced into an existing helix in calmodulin. The crystalstructure of calmodulin reveals an exposed [alpha]-helix wheresubstitutions could be made without altering the amino acid residuesresponsible for calcium binding. In comparison, fusion of calmodulin(147 amino acids) to GFP (238 amino acids) would form a chimericpolypeptide more than two and a half times larger than calmodulin alone.Such a large increase in size might perturb the biological activity orlocalization of calmodulin.

Four cysteines were introduced into the N-terminal α-helix of Xenopuscalmodulin as shown below (SEQ ID NOS:6 and 7):

           5   6   7   8   9   10  11  12  13wild type: Thr Glu Glu Gln Ile Ala Glu Phe Lys mutated:    -  Cys Cys  —  —  Cys Cys  —   —The mutated calmodulin is referred to as calmodulin+cys4. Thesubstitutions were generated by using as a PCR primer of the followingoligonucleotide sequence 5′-CGC GGA TCC GCC ACC ATG CAT GAC CAA CTG ACATGC TGC CAG ATT TGC TGC TTC AAA GAA GCC TTC TCA TTA TTC-3′-(SEQ ID NO.3) encoding for the expression of these substitutions. The nucleic acidsequence encoding the cysteine-substituted calmodulin was inserted intopcDNA3 vector (Invitrogen, Carlsbad, Calif.) using the BamHI and EcoRIrestriction sites. After amplification in DH5 bacteria, the vector wastransfected (at 37° C.) into HeLa cells using the Lipofectin system fromGibcoBRL.

Three days after transfection, the cells were treated with 1 μMbiarsenical molecule (III) and 10.0 μM EDT for one hour. Observation ona fluorescence microscope stage using a 480DF30 filter (Omega Optical,Brattleboro, Vt.) and a 4% transmittance neutral density filter forexcitation and a 535DF25 filter for emission revealed many cells withbright fluorescence compared to adjacent lightly stained cells. Theselightly stained cells may have expressed the calmodulin+cys4polypeptide, but at lower levels than the bright cells. Untransfectedcells treated with the same concentrations of biarsenical molecule (III)and EDT had only very light fluorescence. Removal of the 1.4 neutraldensity filter was required to see details of the staining of theuntransfected cells which appeared to be mitochondrial. This experimentdemonstrated the feasibility of using biarsenical molecule (III) tolabel polypeptides within cells by creating a target sequence intoalready existing polypeptides, leaving the molecular weight of thepolypeptide essentially unchanged.

Example 4 Synthesis of Dichloro Derivative of Biarsenical Molecule (III)

A solution of 84 mg (265 μmol) mercuric acetate in 500 μl 1:1 aceticacid/water was added (at room temperature) to a solution of 19 mg (47μmol) of 2′,7′-dichlorofluorescein in 500 μl ethanol. After stirringovernight, the red solid was filtered, rinsed with ether and dried undervacuum. 20 mg (22 μmol, 47%) of2′,7′-dichloro-4′,5′-di(acetoxymercuri)fluorescein was collected.

The dichloro derivative of biarsenical molecule (III)(2′,7′-dichloro-4′,5′-bis(2-arsa-1,3-dithiolan-2-yl)fluorescein) wasprepared as follows. 2′,7′-dichloro-4′,5′-di(acetoxymercuri)fluorescein(13 mg, 14 μmol) was prepared as described above and suspended in 500 μldry N-methylpyrrolidinone. Upon addition of 24 μl (285 μmol) arsenictrichloride, the suspended solid dissolved to a light yellow solution.DIEA (20 μl) and a catalytic amount of palladium diacetate were added.After three hours, the dark reaction mixture was quenched with 2.5 ml1:1 acetone/water. EDT (200 μl) was added and the reaction stirred for45 minutes. The product was extracted into chloroform. The organic layerwas washed with saturated NaCl. Most of the solvent was removed byrotary evaporation and then additional chloroform was added. The whitesolid that precipitated was discarded. The product was isolated onsilica gel with ethyl acetate-hexane 1:1, 0.1% acetic acid as eluant.The retention factor, R_(f) was 0.6 (1:1 ethyl acetate-hexane 1:1, 0.1%acetic acid). The yield was 113 nmole (1%) as determined by absorbanceassuming a peak extinction coefficient of 80,000 M⁻¹ cm⁻¹.

Formation of a complex with target sequence and the dichloro-derivativeof the biarsenical molecule (III). Dichloro-derivative of thebiarsenical molecule (III) (5 μl of 675 μm solution in DMSO was added to100 μl of 25 μM target sequence (SEQ ID NO. 1) in 25 mM phosphate, pH7.4, 100 mM KCl 1 mM mercaptoethanesulfonate. After 1.5 hours, thereaction mixture was injected onto a Dionex IonPac SN1 reverse phaseHPLC column, gradient 20% to 46% acetonitrile (0.1% TFA) from 3 to 17minutes. The complex eluted in two overlapping peaks of the samemolecular weight at 16.1 and 16.4 minutes. (Free peptide elutes at 12.6minutes.) Mass spectrum analysis indicated a molecular weight of 2484.80Da compared to the calculated molecular weight of the 1:1 complex was2484.22 Da.

The dichloro-derivative of the biarsenical molecule (III) behavedsimilarly to the biarsenical molecule (III). A 10 nm red shift wasobtained (excitation at 518 nm, emission at 538 nm).

Example 5 Synthesis of Tetraarsenical Molecules

Bifluorescein molecule. (See also O Silberrad (1906). J. Chem. Soc., 89,1787-1811 and S. Dutt (1926), J. Chem. Soc., 1926, 1171-1184).Pyromellitic acid (744 mg, 2.93 mmol) and resorcinol (1.367 g, 12.4mmol) were heated at 160° C. for two hours in the absence of solvent.After cooling to room temperature, the solid product was boiled in waterand filtered. The orange solid that was collected was suspended inethanol and filtered. 196 mg of crude product was precipitated uponaddition of water to this filtrate. Two products giving closely spacedTLC spots (R_(f)=0.08 1:1 ethyl acetate/hexane) were isolated on silicagel with 99.9% ethyl acetate, 0.1% acetic acid. These most likely arethe para- and meta-substitution isomers (FIG. 2). The orange solid (84mg) containing the two isomers was collected (5%). Mass spectrumanalysis indicated a molecular weight of 586.47 and the calculatedmolecular weight was 586.51.Tetrakis(acetoxymercuri)bifluorescein. Mercuric acetate (252 mg, 790μmol) dissolved in 2 mL 1:1 water/acetic acid was added to a mixture of84 mg (143 μmol) bifluorescein in 6 ml ethanol. After stirringovernight, all material remained on baseline by TLC (1:1ethylacetate/hexane) indicating that mercuration had occurred. The darkred solid (149 mg, 64%) collected by filtration was not furthercharacterized.Tetraarsenical molecules. The above material (67 mg, 41 μmol) suspendedin 3 mL dry N-methylpyrrolidinone dissolved to a yellow solution uponaddition of 140 μl (1.66 mmol) AsCl₃. DIEA (290 mL) and a catalyticamount of palladium diacetate were added. After 1.5 hours, the reactionwas poured into a mixture of 5 mL acetone, 5 mL pH 7.4 phosphate bufferand 2 mL EDT. After removing the solvent from a chloroform extract ofthe aqueous reaction, the product was isolated on silica gel with 1:1ethylacetate/hexane 0.1% acetic acid. Mass spectral analysis indicated amolecular weight of 1250.47 Da compared to a calculated molecular weightof 1250.86.

The final product isolated on silica was mostly of the correct mass(mass spectrum analysis indicated a molecular weight of 1250.47 and thecalculated molecular weight was 1250.86). A small peak was also presentcorresponding to the mass of a product with one arsenic group missing(mass spectrum analysis indicated a molecular weight of 1084.55 and thecalculated molecular weight was 1084.75.)

Formation of the tetraarsenical molecule complex with two targetsequences. Tetraarsenical molecule (5 μl of 550 μM in DMSO) and 3 μl of1.4 mM target sequence (SEQ ID NO. 1) were added to 50 μl 25 mMphosphate, pH 7.4, 100 mM KCl, 1 mM mercaptoethanesulphonate. After 1.5hours, 10 μl of the reaction mixture was injected onto a C-18 reversephase HPLC column linked to a mass spectrometer. A peak that eluted at13 minutes contained a species with molecular weight of 4752.07 Da,compared to the calculated molecular weight for the 2:1 complex of4752.11 Da, indicating that the desired complex had been formed.

Other embodiments are within the following claims. For example, thebiarsenical molecule can have the following formula

One specific embodiment can have the following formula

1. A kit comprising a. a biarsenical molecule of the formula:

and tautomers, anhydrides, and salts thereof; and b. a bonding partnercomprising a target sequence and a carrier molecule, said targetsequence comprising four cysteines capable of specifically reacting withsaid biarsenical molecule.
 2. The kit of claim 1, wherein said targetsequence comprises a Cys-Cys-X-Y-Cys-Cys sequence, wherein said X and Yare amino acids.
 3. The kit of claim 1, wherein said target sequence isselected from the group consisting of SEQ ID NO. 1 and SEQ ID NO.
 4. 4.The kit of claim 1, wherein said carrier molecule comprises a carrierpolypeptide.
 5. The kit of claim 4, wherein said target sequence isheterologous to said carrier polypeptide.
 6. The kit of claim 1, whereinsaid biarsenical molecule specifically reacts with said target sequenceand generates a detectable signal.
 7. The kit of claim 6, wherein saiddetectable signal is a fluorescent signal.
 8. A kit comprising a. abiarsenical molecule of the formula:

and tautomers, anhydrides, and salts thereof; and b. a vector comprisinga nucleic acid sequence encoding a target sequence, said nucleic acidsequence operatively linked to a promoter sequence and said targetsequence comprising one or more cysteines capable of specificallyreacting with said biarsenical molecule.
 9. The kit of claim 8, whereinsaid target sequence comprises four cysteines.
 10. The kit of claim 8,wherein said target sequence is selected from the group consisting ofSEQ ID NO. 1 and SEQ ID NO.
 4. 11. The kit of claim 8, wherein saidvector further comprises a nucleic acid sequence encoding a carrierpolypeptide.
 12. The kit of claim 11, wherein said carrier polypeptideis heterologous to said target sequence.
 13. The kit of claim 8, whereinsaid biarsenical molecule specifically reacts with said target sequenceand generates a detectable signal.
 14. The kit of claim 13, wherein saiddetectable signal is a fluorescent signal.
 15. A complex comprising abiarsenical molecule and a target sequence associated with a carrierpolypeptide, said target sequence comprising one or more cysteinescapable of reacting with said biarsenical molecule, said biarsenicalmolecule having the following formula:

and tautomers, anhydrides, and salts thereof.
 16. The complex of claim15, wherein said target sequence comprises four cysteines.
 17. Thecomplex of claim 15, wherein said target sequence is selected from thegroup consisting of SEQ ID NO. 1 and SEQ ID NO. 4.